Fig 2: The XPC-M region regulates the subnuclear localization of XPC(A) Deletion mutants of human XPC used in this study. Positions of the transglutaminase-homology domain (TGD) and three ß-hairpin domains (BHD1/2/3) are indicated.(B) The wild-type and mutant XPC proteins in (A) were fused to the FLAG-EGFP tandem tag and stably expressed in Xpc-deficient MEFs. Immunoblot confirming the protein expression is shown.(C) The cells used in (B) were fixed, counterstained with Hoechst 33342, and observed under a confocal laser scanning fluorescence microscope. Scale bar: 5 µm.(D) Correlation coefficients between EGFP and Hoechst 33342 signals were calculated and plotted for individual nuclei using images from (C). Two independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. The median with 95% confidence interval is shown for each sample. Statistical analysis was carried out by Kruskal-Wallis test followed by Dunnett's test, and only the results among WT, ?M, and NLS-M are shown. ****p < 0.0001.

Fig 3: MTA proteins regulate histone deacetylation and XPC recruitment at LUD sites(A) Three MTA proteins, HDAC2, and HDAC3 fused to HaloTag 7 were individually expressed in DDB2-deficient U2OS cells expressing EGFP-XPC. After labeling with the HaloTag TMR ligand, the cells were irradiated with UVC (400 J/m2) through isopore membrane filters. After incubation for 15 min, the cells were fixed and observed under a confocal laser scanning microscope. Scale bar: 5 µm.(B) Cells expressing the indicated proteins fused to HaloTag 7 were treated as shown in (A). Relative TMR intensity in each irradiated area (identified by EGFP-XPC accumulation), in comparison with the entire nucleus, was calculated and plotted. The mean with 95% confidence interval is shown for each sample. Two or three independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. One sample t-test (two tailed) was used to assess whether the mean value of each sample was greater than 1. p value: 3.9E-20 (MTA1), 2.9E-07 (MTA2), 4.6E-10 (MTA3), 2.6E-05 (HDAC2), 1.0E-04 (HDAC3).(C) Laser-based local UVC stimulation was applied to cells co-expressing Halo-MTA1 and EGFP-XPC. Fluorescent images were acquired before and 150 s after stimulation. Scale bar: 5 µm.(D) Indicated proteins fused to HaloTag 7 were ectopically expressed in DDB2-deficient U2OS cells expressing EGFP-XPC. After local UVC stimulation was applied as in (C), the accumulation of TMR fluorescence was monitored by time-lapse imaging. The TMR intensity in each stimulated area relative to the entire nucleus was calculated and plotted over time (the graph shows the means and SDs at each time point). Three independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. Endpoint data were subjected to one-way ANOVA followed by Tukey's HSD test, and statistical significance compared with HDAC3 is shown. ****p < 0.0001. See also Figure S6A.(E) Immunoblot confirming the depletion of individual MTA proteins 3 days post siRNA treatment.(F) DDB2-deficient U2OS cells expressing EGFP-XPC were treated with the indicated siRNAs for 3 days. Upon local UVC stimulation, the accumulation of EGFP-XPC was monitored. The EGFP intensity in each stimulated area relative to the entire nucleus was calculated and plotted (the graph shows the means and SDs at each time point). Three independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. Statistical significance compared with siNC is shown, based on the analysis in Figure S6C. ****p < 0.0001.(G) The three MTA proteins were simultaneously depleted in DDB2-deficient U2OS cells expressing EGFP-XPC. Local UVC irradiation (400 J/m2) was performed through isopore membrane filters, and relative levels of H3K27ac in the irradiated areas were assessed as in Figure 1E. Two independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. Student’s t test (two tailed) was used for statistical analyses. ****p < 0.0001.

Fig 4: XPC localizes to hypoacetylated chromatin regions(A) EGFP-XPC and DsRed-HP1a were simultaneously expressed in Xpc-deficient MEFs. The cells were treated for 6 h with 0 or 2 µM TSA, fixed, counterstained with DAPI, and observed under a fluorescence microscope. Scale bar: 10 µm (wide-field image), 5 µm (enlarged image).(B) Xpc-deficient MEFs expressing EGFP-XPC were fixed and stained with anti-H3K27ac antibody. Scale bar: 10 µm (upper panel), 5 µm (lower panel).(C) Schematic illustration of the LacO-LacR tethering system.(D) Cells harboring lacO arrays and stably expressing EGFP-XPC were transfected with constructs for the expression of HDAC2-mCherry-LacR, HDAC1-mCherry-LacR, or mCherry-LacR alone, and incubated for 2 days. After pre-extraction and fixation, the cells were observed under a confocal laser scanning fluorescence microscope. Representative images of transfected cells are shown. Scale bar: 5 µm.(E) Relative EGFP intensity at the lacO arrays (marked by mCherry fluorescence) in comparison with the entire nucleus was calculated and plotted using fluorescent images from (D). HDAC3-mCherry-LacR was also included in this quantitative analysis. Two independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. The median with 95% confidence interval is shown for each sample. Statistical difference from control (mCherry-LacR only) was assessed by Kruskal-Wallis test followed by Dunnett's test. ****p < 0.0001.(F and G) Cells stably expressing EGFP-XPC were treated with siNC or a mixture of siRNAs targeting HDAC1 and HDAC2 (siHDAC1/2). Subsequently, mCherry-LacR fused to siRNA-resistant (siR) wild-type (D) or H142A mutant HDAC2 (E) was transiently expressed. mCherry-LacR alone was used as a control. Relative EGFP intensity at the lacO arrays in comparison with the entire nucleus was calculated and plotted as in (E). Two independent experiments were performed, and the total number of cells analyzed (n =) for each condition is shown in gray. Statistical analysis was performed by Kruskal-Wallis test followed by Dunnett's test. ***p < 0.001, ****p < 0.0001.

Fig 5: Roles for HDACs in UV-DDB-independent DNA lesion recognition by XPC(A and B) DDB2-deficient U2OS cells stably expressing EGFP-XPC were pre-treated for 6 h with various concentrations of TSA (A) or romidepsin (B). Local UVC stimulation was applied, and the accumulation of EGFP-XPC to LUD sites was monitored by time-lapse imaging. The EGFP intensity in each stimulated area relative to the entire nucleus was calculated and plotted as described previously (Sakai et al., 2020). The graphs show the mean and SDs at individual time points (32 cells were analyzed for each condition in two independent experiments). Statistical significance compared with control samples (without TSA or romidepsin) is shown, based on the analyses in Figure S1C. *p < 0.05, ****p < 0.0001.(C) Immunoblot confirming the depletion of individual class I HDACs 2 days post siRNA treatment. siNC: negative control siRNA.(D) DDB2-deficient U2OS cells expressing EGFP-XPC were treated for 2 days with the indicated siRNAs. The accumulation of EGFP-XPC was monitored over time following local UVC stimulation and plotted as in (A and B). Two or three independent experiments were performed for each condition and the total number of cells analyzed (n =) is shown in gray. Statistical significance compared with siNC is shown based on the analysis in Figure S2A. **p < 0.01.(E) DDB2-deficient U2OS cells expressing EGFP-XPC were irradiated with UVC (400 J/m2) through isopore membrane filters. After 30 min incubation, immunofluorescence staining was carried out with the anti-H3K27ac antibody. Relative intensity of the H3K27ac stain in each irradiated area (identified by EGFP-XPC accumulation), in comparison with the entire nucleus, was calculated and plotted. Two independent experiments were performed, and the total number of cells analyzed (n =) is shown in gray. The mean with 95% confidence interval is shown for each sample. Statistical analysis was performed by one-way ANOVA followed by Tukey's HSD test. **p < 0.01.(F) Either HDAC2 or HDAC3 fused to HaloTag 7 was ectopically expressed in DDB2-deficient U2OS cells expressing EGFP-XPC. After the cells were incubated for 30 min in the presence of the HaloTag TMR ligand, local UVC stimulation was applied to monitor the accumulation of HaloTag 7-fusion proteins. The TMR intensity in each stimulated area relative to the entire nucleus was calculated and plotted over time (the graph shows the means and SDs at each time point). Three independent experiments were performed and the total number of cells analyzed (n =) is shown in gray. Statistical analysis of endpoint data was performed by Student’s t test (two tailed). ****p < 0.0001. See also Figure S6A.